99mTc-labeled duramycin as a novel phosphatidylethanolamine-binding molecular probe
99mTc-labeled duramycin as a novel phosphatidylethanolamine-binding molecular probe
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DOI：
10.2967/jnumed.107.048603
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发表时间：
2008-08-01
影响因子：
9.3
通讯作者：
Bugenhagen, Scott
中科院分区：
文献类型：
--
作者：
Zhao, Ming;Li, Zhixin;Bugenhagen, Scott
只有19个氨基酸，duramycin是已知的最小的多肽，具有明确的三维结合结构。Duramycin以1：1的比例与磷脂酰乙醇胺（PtdE）结合，具有高亲和力和排他性特异性。PtdE作为一种丰富的结合靶点，是哺乳动物细胞膜中主要的磷脂，约占磷脂含量的20%。PtdE外化到凋亡细胞的表面，并且由于质膜完整性受损而在坏死细胞中也变得可接近。鉴于duramycin独特的理化性质和PtdE在急性细胞死亡中的可用性，本研究的目的是开发和评价Tc-99 m-duramycin作为PtdE成像的新型分子探针。研究方法：用琥珀酰亚胺基6-肼基烟酸丙酮腙（HYNIC）共价修饰Duramycin，并使用涉及三羟甲基甘氨酸-膦共配体的配位化学用Tc-99 m标记。保留的PtdE结合活性，证实了使用竞争测定与含PtdE的脂质体。测定了Tc-99 m-duramycin在大鼠体内的血液清除率、药代动力学和生物分布。最后，Tc-99 m-duramycin结合急性细胞死亡在体内被证明使用大鼠模型的急性心肌梗死诱导的缺血和再灌注，并确认使用放射自显影和组织学。结果：合成了化学计量比为1：1的HYNIC衍生化的度霉素，并经质谱确证。放射性标记效率为80%~ 85%，放化纯度为78%~ 89%，比活度为54 GBq。放射性示踪剂在使用前用高效液相色谱放射检测器纯化。凋亡细胞对Tc-99 m-duramycin的特异性摄取比对照细胞增加30倍以上。这种结合在含PtdE的脂质体的存在下竞争性地减少，但不是由其他磷脂物质组成的脂质体。静脉注射Tc-99 m-duramycin具有良好的药代动力学和生物分布特征：它通过肾脏系统从循环中快速清除，在大鼠中的血液半衰期小于4分钟。肝脏和胃肠道摄取非常低。Tc-99 m-耐久霉素在体内完全不代谢，从尿液中回收完整的药物。结合快速清除和低肝脏背景，Tc-99 m-duramycin与梗死心肌的亲和结合在注射后不久迅速变得明显。通过放射自显影和组织学证实了梗死组织中的放射性摄取。结论：Tc-99 m-duramycin是一种稳定的低分子量PtdE结合放射性药物，具有良好的体内显像特性。它是一个强有力的候选人作为PtdE成像的分子探针，并保证进一步的发展和表征。
With only 19 amino acids, duramycin is the smallest known polypeptide that has a defined 3-dimensional binding structure. Duramycin binds phosphatidylethanolamine (PtdE) at a 1:1 ratio with high affinity and exclusive specificity. As an abundant binding target, PtdE is a major phospholipid and accounts for about 20% of the phospholipid content in mammalian cellular membranes. PtdE is externalized to the surface of apoptotic cells and also becomes accessible in necrotic cells because of compromised plasma membrane integrity. Given the unique physicochemical properties of duramycin and the availability of PtdE in acute cell death, the goal of this study was to develop and evaluate Tc-99m-duramycin as a novel molecular probe for imaging PtdE. Methods: Duramycin is covalently modified with succinimidyl 6-hydrazinonicotinate acetone hydrazone (HYNIC) and labeled with Tc-99m using a coordination chemistry involving tricine-phosphine coligands. The retention of PtdE-binding activities was confirmed using competition assays with PtdE-containing liposomes. The blood clearance, pharmacokinetics, and biodistribution of Tc-99m-duramycin were measured in rats. Finally, Tc-99m-duramycin binding to acute cell death in vivo was demonstrated using a rat model of acute myocardial infarction induced by ischemia and reperfusion and confirmed using autoradiography and histology. Results: HYNIC-derivatized duramycin with 1:1 stoicheometry was synthesized and confirmed by mass spectrometry. The radiolabeling efficiency was 80%-85%, radiochemical purity was 78%-89%, and specific activity wets 54 GBq. The radiotracer was purified with high-performance liquid chromatography radiodetection before use. The specific uptake of Tc-99m-duramycin in apoptotic cells, compared with that in viable control cells, was enhanced by more than 30-fold. This binding was competitively diminished in the presence of PtdE-containing liposomes but not by liposomes consisting of other phospholipid species. Intravenously injected Tc-99m-duramycin has favorable pharmacokinetic and biodistribution profiles: it quickly clears from the circulation via the renal system, with a blood half-life of less than 4 min in rats. The hepatic and gastrointestinal uptake were very low. Tc-99m-duramycin is completely unmetabolized in vivo, and the intact agent is recovered from the urine. Combined with a fast clearance and low hepatic background, the avid binding of Tc-99m-duramycin to the infarcted myocardium quickly becomes conspicuous shortly after injection. The uptake of radioactivity in infarcted tissues was confirmed by autoradiography and histology. Conclusion: Tc-99m-duramycin is a stable, low-molecular-weight PtdE-binding radiopharmaceutical, with favorable in vivo imaging profiles. It is a strong candidate as a molecular probe for PtdE imaging and warrants further development and characterization.